过表达Abba1重组表达质粒对COS7细胞膜结构影响的实验研究

发布时间：2018-01-14 20:11

  本文关键词：过表达Abba1重组表达质粒对COS7细胞膜结构影响的实验研究　出处：《南京医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 细胞膜结构 Abba1 IMD ruffling Rac1

【摘要】：背景和目的:细胞运动是人类生命活动中重要的病理生理基础,与衰老、血管新生和肿瘤转移密切相关。细胞膜结构重塑是细胞运动的重要结构基础,主要包括胞膜突起、凹陷和管状结构的形成。在胞膜突起结构中根据形态和直径大小可以大致分为microspikes(微刺状)和ruffling(皱褶状)结构。目前的研究认为,IMD (IRSp53/MIM homology domain)蛋白质家族参与细胞膜结构重塑。该蛋白质家族共有一段高度保守的IMD结构域,其中一个家族成员是Abba1。体外实验中将Abba1和Abba1-IMD分别转染细胞,发现过表达全长的Abba1主要促进细胞ruffling生成,而转染单独的IMD主要促进细胞microspikes的生成。本实验旨在初步探讨Abba1氨基酸序列中调节细胞膜结构重塑的结构域。 方法:本实验将全长的hAbba1 (human Abba1,人源Abba1)序列或hAbba1的序列片段分别载入表达GFP绿色荧光蛋白的表达载体pEGFP中,形成Abba1的基因重组表达质粒,形成Abba1的重组表达质粒。 包括: 4177(pEGFP-hAbba1-del-WH2) 4178(pEGFP-hAbba1-IMD) 4210(pEGFP-hAbba1-del-SRD) 4764(pEGFP-hIRSp53-IMD-hAbba1-SRD1-SRD2-WH2) 4842(pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like)5160(pEGFP-hAbba1-IMD-(Lys13Glu13)-SRD1-SRD2-WH2) 5244(pEGFP-hAbba1-IMD-SRD1-SRD2-(SerAla)-WH2) 5231(pEGFP-hAbba1-del-357-553aa-WH2) 5232(pEGFP-hAbba1-del-357-722aa-WH2) 5399(pEGFP-hAbba1-del-554-722aa-WH2)。 双酶切和基因测序验证上述Abba1基因重组表达质粒。将制备成功的重组表达质粒分别瞬时转染COS7细胞,通过免疫荧光将GFP绿色荧光蛋白染色从而示踪显示转染成功的COS7细胞,并在显微镜下观察细胞膜结构的改变。 结果:免疫荧光结果显示:同GFP对照组相比,过表达全长的Abba1主要促进细胞ruffling生成,与转染全长Abba1产生类似的细胞膜突起结构的基因重组表达质粒还有4764(pEGFP-hIRSp53-IMD-hAbba1-SRD1-SRD2-WH2), 4177(pEGFP-hAbba1-del-WH2),4210(pEGFP-hAbba1-del-SRD),5244(pEGFP-hAbba1-IMD-SRD1-SRD2-(SerAla)-WH2),5231(pEGFP-hAbba1-del-357-553aa-WH2), 5399(pEGFP-hAbba1-del-554-722aa-WH2);与转染全长Abba1的结果相反,过表达5160(pEGFP-hAbba1-IMD-(Lys13Glu13)-SRD1-SRD2-WH2)的COS7细胞基本不形成ruffling;比较特殊的是转染4178(pEGFP-hAbba1-IMD)的COS7细胞基本不形成ruffling,而是主要促进细胞microspikes的生成。类似的结果还发生在过表达基因重组表达质粒4842(pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like)和5232(pEGFP-hAbba1-del-357-722aa-WH2)的COS7细胞中。 研究证实Abba1的IMD能与Rac1可以相互作用,并通过上调GTP-Rac1(Rac1的活性形式)的蛋白水平促进细胞产生ruffling。本实验发现,与对照组相比,过表达全长Abba1的COS7细胞中GTP-Rac1蛋白水平升高(P 0.05),而过表达4178(pEGFP-hAbba1-IMD)和5232(pEGFP-hAbba1-del-357-722aa-WH2)的COS7细胞中GTP-Rac1蛋白水平降低(P 0.05)。 结论:Abba1单独的IMD主要促进细胞microspikes的生成。在全长的Abba1序列中,IMD又参与调节细胞产生ruffling,其13个赖氨酸为主要的功能位点。因此IMD是调节不同细胞膜结构重塑的重要功能域。Abba1的357-722aa这段氨基酸序列对细胞ruffling的产生也很关键,而且实验结果提示该区域参与Abba1介导细胞产生ruffling的功能位点可能跨越357-722aa的整个区域。初步的蛋白水平研究发现357-722aa这段区域参与Abba1介导细胞产生ruffling与GTP-Rac1蛋白水平的下调有关。
[Abstract]:BACKGROUND & OBJECTIVE : Cell motility is an important pathological and physiological basis in human life activities . It is closely related to aging , angiogenesis and tumor metastasis . The membrane structure remodeling is an important structural foundation of cell movement . It is considered that the protein family of IMD ( IRSp53 / MIM homology domain ) is involved in the remodeling of cell membrane structure . In the present study , the family member is Abba1 . In vitro experiments , Abba1 and Abba1 - IMD are transfected into cells . Methods : The whole length of hAbba1 ( human Abba1 , human Abba1 ) or hAbba1 sequence fragment was loaded into the expression vector of GFP green fluorescent protein , and the recombinant expression plasmid of Abba1 was formed , and the recombinant expression plasmid of Abba1 was formed . It includes 4177 ( - hAbba1 - del - WH2 ) 4178 ( HAbba1 - del - WH2 ) 4178 ( HIRSp53 - IMD - hAbba1 - SRD1 - SRD2 - WH2 ) 4842 ( - hAbba1 - IMD - hAbba1 - SRD1 - SRD2 - WH2 ) 5231 ( - hAbba1 - del - 357 - 553aa - WH2 ) 5232 ( - hAbba1 - del - 357 - 722aa - WH2 ) 5399 ( - hAbba1 - del - 554 - 722aa - WH2 ) . The recombinant expression plasmid of the above - mentioned Abba1 gene was verified by double digestion and gene sequencing . The recombinant expression plasmids were transiently transfected into COS7 cells , and GFP green fluorescent protein was stained by immunofluorescence to show the successful COS7 cells , and the changes of cell membrane structure were observed under the microscope . Results : The results showed that , compared with the GFP control group , the total length Abba1 promoted the formation of ruminal , and the gene recombinant expression plasmid with similar membrane bulge structure with the transfected full - length Abba1 did not form ruminal , but rather the COS7 cells transfected with the transfected full - length Abba1 did not form ruminal , but it was more specific that the COS7 cells transfected with 4178 ( HAbba1 - IMD - ( Lys 13Glu13 ) - SRD1 - SRD2 - WH2 ) did not form ruminal , but the results of similar results also occurred in COS7 cells expressing the recombinant expression plasmid 4842 ( antisense - hAbba1 - IMD - hIRSp53 - SH3 - WH2 - like ) and 5232 ( - hAbba1 - del - 357 - 722aa - WH2 ) . It was found that the level of GTP - Rac1 protein in COS7 cells expressing full - length Abba1 increased ( P0.05 ) , and the level of GTP - Rac1 protein in COS7 cells expressing 4178 and 5232 was significantly lower than that of control group ( P 0.05 ) . Conclusion : Abba1 alone IMD mainly promotes the formation of microvesicles . In the full - length Abba1 sequence , IMD is involved in regulating the production of ruminal and 13 lysine as the main functional sites . Therefore , IMD is the important functional area for regulating the remodeling of different cell membrane structures . The results suggest that the region is involved in the formation of rumination by Abba1 . The preliminary protein level study found that the region involved in Abba1 - mediated cell production ruminal is related to the downregulation of the level of GTP - Rac1 protein .

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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